|Cray, jr., William|
Submitted to: Journal of Food Protection
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/23/2012
Publication Date: 5/1/2012
Citation: Medina, M.B., Shelver, W.L., Fratamico, P.M., Fortis, L., Narang, N., Cray, Jr., W., Esteban, E., Tillman, G., Debroy, C. 2012. Latex agglutination assays for detection and of non-O157 Shiga toxin-producing E. coli serogroups O26, O45, O103, O111, O121 and O145. Journal of Food Protection. 75(5):819-826. Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens responsible for worldwide outbreaks of hemorrhagic colitis (bloody diarrhea) and hemolytic uremic syndrome (kidney injury). A non-instrumental, rapid, simple and inexpensive agglutination method was developed for the detection of the “big six” non-O157 STEC, namely, the O26, O45, O103, O111, O121, and O145 E. coli. The test consisted of placing a droplet of specific latex-antibody-latex reagent on a glass slide and mixed with E. coli cell colonies. Positive results were shown by agglutination (clumping) occurring within 10 sec. Over 2000 tests were carried out for the six latex-antibody reagents with over 100 E. coli and non-E. coli bacteria and results indicated reliable recognition of target STECs. The agglutination assays are useful for confirming presumptive positive non-O157 STEC colonies, thus greatly enhancing food safety. This test is less costly and less time consuming than testing colonies by polymerase chain reaction (PCR) assays.
Technical Abstract: Latex agglutination assays were developed for the top six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups utilizing polyclonal antibodies. Rabbit antisera were affinity purified through Protein A/G columns and the isolated immunoglobulins (IgG) were covalently immobilized onto polystyrene latex particles. The resulting latex-IgG complex had a protein (IgG) load of 2.0 – 2.8ug/ml in a 20 ml latex suspension containing 1% solids. Optimum conditions for the agglutination assay consisted of utilizing 20 ul of latex-IgG reagent containing 2.0 – 2.8 ng IgG in a 0.5% latex suspension. Agglutination or flocculation was observed almost instantly (less than 10 s) with positive strains against the six serogroups. A total of 106 strains (target and non-target) were tested in over 2000 assays. All target organisms tested positive, except for one strain. Three antisera, anti-O26, O103 and O145 showed cross-reactions with some other STECs belonging to the target serogroups. Of the non-target strains, two showed positive reactions to one of the six antisera. The latex-IgG reagents are stable for at least 6 months and are easy to prepare. These agglutination assays can be used for monitoring of non-O157 STEC serogroups and to confirm presumptive positive colonies from agar media. The method can be used to produce latex reagents for testing of additional STEC serogroups, other E. coli or other pathogens to provide safe foods to consumers.